STEREOCHEMICAL PROBE FOR THE MECHANISM OF P-C BOND-CLEAVAGE CATALYZED BY THE BACILLUS-CEREUS PHOSPHONOACETALDEHYDE HYDROLASE

The enzyme, the phosphonoacetaldehyde hydrolase (phosphonatase) of Bacillus cereus, catalyzes the conversion of phosphonoacetaldehyde to phosphate and acetaldehyde. Previous studies have shown that phosphonase labilizes the C-P bond in the substrate by forming a protonated Schiff base between an active site lysine and the aldehyde carbonyl group (Olsen, B. H.; Hepburn, T. W.; Moos, M.; Mariano, P. S.; Dunaway-Mariano, D. Biochemistry 1988, 27, 2229). In this article, we describe the synthesis of stereochemical probes of this C-P bond cleavage reaction. The enantiomers of the chiral [O-17, O-18] (thiophosphono)acetaldehyde have been prepared for this purpose, and an analysis of the stereochemistry of their phosphonatase-catalyzed transformations to [O-16, O-17, O-18]thiophosphate has been carried out. The synthesis of the enantiomers of [O-17, O-18](thiophosphono)acetaldehyde centered about the preparation and HPLC separation of diastereomeric thiophosphonamide precursors. The absolute phosphorus configurations in the thiophosphonamides were determined by X-ray analysis of a crystalline derivative of the (S(P),S(C) diastereomer. The stereochemistry of the phosphonatase-catalyzed reactions of the chiral (thiophosphono)acetaldehydes was determined to be retention at phosphorus. The results are interpreted in terms of a mechanism involving P-C bond cleavage in a protonated Schiff base intermediate by in-line displacement by an enzyme nucleophile. Subsequent hydrolysis of the resultant acetaldehyde enamine and phosphoenzyme groups then yields acetaldehyde and phosphate.